Our long-term goal is to identify the molecular interactions involved in pancreas-specific gene expression and transcriptional regulation by insulin. Expression of pancreatic amylase is repressed in diabetic animals, reducing absorption of dietary carbohydrate, and re-expressed after treatment with insulin. We have identified a 63 bp DNA segment from the 5' flanking region of a murine pancreatic amylase gene which contains an insulin-responsive element and a pancreas-specific enhancer. Several proteins from pancreatic nuclei bind specifically to this fragment. We will localize the nucleotides essential for insulin response and enhancer activity by functional assays of synthetic mutant enhancer elements with 3 bp substitutions. Band shift assays with each mutant will localize the binding sites for each of the nuclear proteins. By correlating loss of binding with loss of function, we will identify putative transcriptional regulatory proteins. These proteins will be cloned by screening cDNA libraries with oligonucleotide probes. The structure and developmental expression of positive clones will be studied. We will select mutants of the AR42J pancreatic acinar cell line which are defective individual transcription factors. These lines can by used to test the function of cloned proteins. We will evaluate the activity of a glucocorticoid receptor binding site located at position-17 to +1 of the amylase gene. Interaction between two conserved pancreas consensus elements by insulin will be tested in adipocytes and hepatocytes. The experiments proposed here will contribute to understanding the mechanism of insulin action, and will extend our knowledge of pancreas-specific regulation of mammalian genes.